Cassane diterpenoids with α-glucosidase inhibitory activity from the fruits of Pterolobium macropterum

Two new cassane diterpenoids, 14β-hydroxycassa-11(12),13(15)-dien-12,16-olide (1) and 6′-acetoxypterolobirin B (3), together with a known analogue, identified as 12α,14β-dihydroxycassa-13(15)-en-12,16-olide (2), were isolated from the fruits of Pterolobium macropterum. Compound 1 is a cassane diterpenoid with a Δ11(12) double bond conjugated with an α,β-butenolide-type, whereas compound 3 is a dimeric caged cassane diterpenoid with unique 6/6/6/6/6/5/6/6/6 nonacyclic ring system. The structures of 1 and 3 were characterized by extensive spectroscopic analysis combined with computational ECD analyses. The α-glucosidase inhibitory activity of isolated compounds was evaluated, and compounds 1 and 3 showed significant α-glucosidase inhibitory activity with IC50 values of 66 and 44 μM.


Introduction
Diabetes mellitus is a common metabolic disease that affects how the body uses blood glucose. In 2021, 537 million patients suffered from diabetes worldwide, and the number is feared to increase to 783 million in 2045 [1]. Type 2 diabetes account for the majority of the cases [2]. Currently, inhibition of α-glucosidase, the enzyme responsible for the hydrolysis of carbo- hydrates in the body, is widely used for the management of type 2 diabetes. The agents, such as acarbose, miglitol, and voglibose, can retard the digestion and absorption of dietary carbohydrates [3,4]. Some cassane-type diterpenoids such as pulcherrimin C and 6β-cinnamoyl-7β-hydroxyvouacapen-5α-ol, have been reported to exhibit significant α-glucosidase inhibitory activity [5].
The genus Pterolobium, comprising approximately 10 species distributed widely in Africa, China, and Thailand [6], is flowering shrubs belonging to Fabaceae. There are only four species known in Thailand [7], and some of them have been applied as antihemorrhoid [8]. Some species of this genus have revealed cassane diterpenoids as mainly secondary metabolites, which have shown interesting biological activities such as cytotoxicity and anti-inflammatory activity [9][10][11].

Results and Discussion
The fruits of P. macropterum were extracted using MeOH to give a crude extract. After removal of the organic solvent, the extract was separated by repeated silica gel column chromatography as well as by Sephadex LH-20 to afford two new and one known cassane diterpenoids, identified as 12α,14β-dihydroxycassa-13(15)-en-12,16-olide (2) [18].
The HMBC cross-peaks ( Figure 2) from H-11 (δ H 5.86) to C-10, C-12, and C-13, and from H-15 (δ H 6.03) to C-12, C-13   and C-14 allowed the location of an extended conjugated π-system at C-11 and C-12. Moreover, the downfield shift of C-14 (δ C 72.2) and the HMBC correlation between H 3 -17 and C-14 as well as the appearance of the H 3 -17 as a singlet signal confirmed the connection of a hydroxy group at C-14.
The relative configuration of 1 was characterized by NOESY spectra. In the NOESY experiment (Figure 3), the cross-peak between H-8 and H 3 -20 suggested these protons to be syn oriented. In addition, the cross-peaks between H-5/H-9, H-5/H-7α and H-7α/H 3 -17 suggested H 3 -17 to be α-oriented.
Comparison of the specific rotation was used to establish the absolute configuration of 1. The specific rotation of 1 ( −22 (c 0.01, MeOH)) was similar to the reported data of 2 ( −36 (c 0.01, MeOH); lit. −44 (c 0.05, MeOH)) [18], confirming the same absolute configuration these compounds should be derived from the same biosynthetic pathway. In addition, the ECD spectra of (5S,8R,9S,10R,14S)-1 and its enantiomer were calculated at the B3LYP functional using a TD-DFT method [19]. As illustrated in Figure 4a, the measured ECD curve was compared to the predicted ECD curve of (5S,8R,9S,10R,14S)-1, indicating that the measured and predicted ECD spectra were similar except for a blue-shift in the ECD spectrum. Thus, the structure of 1 was characterized as shown.  ]. Careful analysis of the NMR data indicated the presence of a dimeric cassane-type diterpene skeleton whose NMR spectra resembled those of pterolobirin B [11], an unprecedented caged cassane diterpenoid dimer with unique 6/6/6/6/6/5/6/6/6 nonacyclic ring system. The minor difference was the additional acetoxy group (δ H 2.10) at C-6′. This conclusion was suggested by the HMBC correlations ( Figure 2) from H-6′ to 6′-OCOCH 3 (δ C 170.4) and C-10′ (δ C 36.8), and from H 3 -20′ to C-5′ (δ C 55.1) and C-10′, combined with the spin system CH(5′)-CH(6′)-CH 2  3), C-12′, and C-13′ (δ C 71.3) clearly indicated the two C-C bond linkages of both units through the C-15/C-16′ and C-17/C-15′ bonds. Furthermore, the aforementioned ring structure and functionalities accounting for 13 out of 15 degrees of unsaturation required the presence of two heterocyclic rings in the molecule. The presence of an ester carbonyl signal (δ C 167.0) and a deshielded oxygenated carbon resonance at C-12′ (δ C 104.1) implied the formation of six-membered ring via an ester bond between C-16 and C-12′. In addition, an epoxide moiety at C-13′ and C-14′ was further supported by the downfield shift of C-13′ (δ C 71.1) and C-14′ (δ C 65.4) and the cross-peaks from H 3 -17′/H-15′ to C-13′ and C-14′ in the HMBC spectrum.
In the NOESY experiments of 3 (Figure 3 . From above information, the relative configuration of C-12′ was assigned and supported by the biosynthetic pathway based on a Diels-Alder adduct, thus displaying the same relative configuration found in pterolobirin B [11]. The absolute configuration of 3 was thus elucidated as 5S,6R,8R,9S,10R,15R,5′S,6′R, 8′R,9′S,10′R,12′S,13′R,14′R,15′S,16′S and the measured ECD spectrum (Figure 4b) with the positive at 243 nm and negative at 330 nm CEs, is very well matched with the ECD curve of pterolobirin B [11]. Although, the predicted ECD data is not in good agreement with the measured ECD data. It is noted that the calculation could not completely simulate the experimental results depend on the level of theory and basis set as well as the polarity of solvent. Finally, comparison of the specific rotation was used to establish the absolute configuration. Pterolobirin B showed −72 (c 0.1, CHCl 3 ) [11] and 3 showed −87 (c 0.01, MeOH), which also supports the absolute configuration. Thus, the structure of 3 was assigned as shown.
The isolated compounds were evaluated for their α-glucosidase inhibitory activity [20]. Compounds 1 and 3 exhibited significant α-glucosidase inhibitory activity with IC 50 values of 66 and 44 µM, respectively, which showed stronger inhibitory activity than the positive control, acarbose (IC 50 178 μM). Compound 2 was inactive in this assay, with IC 50 value >200 μM, which suggested that a Δ 11 (12) double bond might be important for the α-glucosidase inhibitory activity.

Experimental General experimental procedures
Optical rotations were measured on a JASCO P-2000 polarimeter in MeOH. The UV spectra were recorded on a PerkinElmer UV-vis spectrophotometer. ECD spectra were acquired on a JASCO J-1500 circular dichroism spectrometer. FTIR spectra were obtained using a PerkinElmer FTS FT-IR spectrophotometer. NMR spectra were obtained on a Bruker NEO 500 MHz NMR Ultra Shield. Chemical shifts are referenced in parts per million (δ) in the deuterated solvents (CDCl 3 ) using TMS as an internal standard. An Agilent 1290 infinity II/ Q-TOFMS mass spectrometer was employed to acquire HRESI-TOF-MS spectra. Column chromatography (CC) was carried out on silica gel 60 (70-230 mesh, Merck, Darmstadt, Germany), and Sephadex LH-20 (GE Healthcare). Thin-layer chromatography (TLC) was performed on silica gel 60 F 254 plates (Merck, Darmstadt, Germany) using precoated aluminum plates for analytical purposes.

Extraction and isolation
The fresh fruits of P. macropterum (0.2 kg) were ground and soaked with MeOH (3 × 2 L) at room temperature for 3 days. The solvent was evaporated under reduced pressure at 40 °C, affording MeOH extract (10.5 g). The extract was subjected to silica gel column chromatography (CC) (70-230 mesh, 2 × 60 cm) eluting with hexanes-acetone (100:0 → 0:100, v/v) to afford 10 fractions (Fr. α-Glucosidase inhibitory assay α-Glucosidase inhibitory activity was performed according to experimental literature with slight modification [20]. α-Glucosidase (0.05 U/mL) and substrate, p-nitrophenyl-α-ᴅglucopyronoside (p-NPG) (1 mM) were dissolved in 0.1 M sodium phosphate buffer (pH 6.9). Fifty μL of sample (1 mg/mL in 10% DMSO) and 50 μL of α-glucosidase were preincubated at 37 °C for 10 min in a 96 well plate. The substrate solution (50 μL) was added to the mixture to start the reaction, with further incubation at 37 °C for 20 min. The reaction was terminated by adding 1 mL of 0.3 M Na 2 CO 3 . Enzymatic activity was quantified by measuring the absorbance at 405 nm. The percent inhibition of activity was calculated as (A 0 − A 1 )/A 0 × 100, where A 0 is the absorbance of control, and A 1 is the absorbance with the sample. Acarbose was used as a standard drug and all experiments were evaluated in triplicate.

Supporting Information
Supporting Information File 1 Copies of NMR spectra for compounds 1 and 3.